Project Summary. Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by progressive deterioration of motor neurons in the CMS. There is no treatment or cure for this disease. SMA is the result of the homozygous mutation or deletion of the SMN1 (survival motor neuron-1) gene which encodes the SMN protein (SMNp) which is necessary for the survival of motor neurons. A nearly identical gene in humans, SMN2, fails to prevent SMA because its pre-mRNA undergoes aberrant splicing resulting in an unstable and non-functional SMNp. Pharmacologic modulation of SMA/2mRNA splicing, resulting in increased expression of full-length SMNp, has been reported for the anthracycline drug class. Paratek Pharmaceuticals proposes to develop a novel tetracycline (TC) compound, which has structural similarity to the anthracycline class, to provide a safe, effective drug for modifying SMN2 splicing and improving motor neuron survival in SMN patients. Previous efforts at Paratek have led to the discovery of a TC having significant splice-modifying activity and which increases the expression of full-length SMN2 mRNA in vitro and in vivo. The goals of the present proposal are to (1) synthesize and characterize new TC derivatives in order to maximize splicemodifying activity, (2) optimize the pharmacokinetic characteristics and in vivo efficacy of these novel TCs, and (3) develop a candidate compound with the ultimate goal of filing an IND for its application as a therapeutic treatment for SMA. Paratek will be collaborating with Adrian Krainer at Cold Spring Harbor Laboratory and Michelle Hastings at Chicago Medical School for this work. The proposed methods will include: (1) testing TC derivatives in a cell-free SMV2mRNA splicing assay; (2) in vitro testing of splice-modifying compounds for increased cellular expression of SMNp; (3) in vivo analysis of PK and brain-penetrating activity of TCs; (4) in vivo testing of selected compounds for increased expression of human S/W/V2mRNA in smn;SMN2 transgenic mice and enhanced survival in smn-/-;SMN2 neonate mice. Compounds having in vivo efficacy will undergo preclinical toxicologic, genotoxic and safety pharmacologic analyses to select a candidate for clinical development. Relevance. The goal is to discover, develop, and bring to clinical trial a tetracycline drug for use in the treatment of spinal muscular atrophy (SMA). The drug is intended to prolong and improve the quality of life for children and adults with SMA, a disease for which there presently is no cure.